Method for predicting the risk of transplant rejection and immunological testkit

ABSTRACT

The invention relates to a method and an immunological test kit for predicting the risk of transplant rejection by detecting anti-AT 1  receptor auto-antibodies in biological materials of a patient to be examined, e.g. by an immune reaction with the AT 1  receptor or functionally analogous peptides or proteins thereof. The invention is also directed to the use of the AT 1  receptor or functionally analogous peptides or proteins in order to predict (diagnose) the risk of transplant rejection. Said risk exists if anti-AT 1  receptor auto-antibodies can be detected in such biological materials, e.g. in body fluids or tissues.

[0001] The invention relates to a method and an immunological test kitfor predicting the risk of transplant rejection by detecting anti-AT₁receptor auto-antibodies in biological materials of a patient to beexamined, e.g. by an immune reaction with the AT₁ receptor orfunctionally analogous peptides or proteins thereof. The invention isalso directed to the use of the AT₁ receptor or functionally analogouspeptides or proteins in order to predict (diagnose) the risk oftransplant rejection. Said risk exists if anti-AT₁ receptorauto-antibodies can be detected in biological materials, e.g. in bodyfluids or tissues.

[0002] For successful organ transplantation, the donor organ is requiredto match histologically with the tissue of the recipient as much aspossible. Such matching is determined via the HLA system (humanleukocyte antigen) which is a complex system of tissue antigensoccurring on virtually any cell. Said system plays an importantphysiological role in immunological defense reactions (recognition of“self” and “non-self”). Prior to each transplantation, therefore, aso-called tissue typing is effected in the organ donor and recipient soas to ensure HLA compatibility as much as possible.

[0003] As a result of the immense genetic polymorphism, there is anexceptionally large number of various HLA molecules. Complete matchsolely is observed in monozygotic twins. Otherwise, HLA molecules areunique to each person.

[0004] However, problems exist in that rejection reactions against thetransplanted organ cannot be ruled out despite extensive HLA matchbetween recipient and donor.

[0005] The object of the invention is therefore to provide an efficientand reliable method allowing positive and rapid predictability as to therisk of a transplant rejection reaction.

[0006] The present invention solves this technical problem by providingsuch a method wherein anti-AT₁ receptor auto-antibodies are detected inbiological materials of a patient to be diagnosed, a risk of transplantrejection being present or predictable in those cases where theseauto-antibodies are detected. According to the invention, it ispreferred to detect the auto-antibodies by immune reaction with the AT₁receptor or with peptides or proteins of analogous function. However, itis also possible to detect the auto-antibodies by means of other methodsper se known to those skilled in the art, e.g. via electrophoreticseparation methods. The invention is based on the surprising evidencethat patients exhibiting a transplant rejection reaction with nopredictable immunological risk have anti-AT₁ receptor auto-antibodies.

[0007] It has been possible to demonstrate that there is a relationshipbetween the presence of said auto-antibodies and transplant rejection.In cytobiological and immunohistochemical studies, as well as ininvestigations on biopsy material, it has been found that there are noadditional immunological risk factors to transplant rejection. On theother hand, it has been demonstrated that the presence of theauto-antibodies gives rise to rejection reactions in a recipient withtransplanted organs.

[0008] In connection with the present invention, a number of generalterms will be used as follows:

[0009] “Transplant” in the meaning of the invention is an organ or atissue which has been transplanted or is to be transplanted. In themeaning of the invention, however, transplants can also be particularimplants comprised of materials or components incorporated in a body fora limited period of time or for life in order to assume specificsubstitute functions. For example, such implants can be made ofinorganic matter coated with organic substances such as cartilage orbone cells.

[0010] According to the invention, “transplant rejection” is understoodto be induction of an immune reaction to the transplant in therecipient. An immune reaction in the recipient is a specific protectiveor defense reaction of the body against the antigens of the transplant.

[0011] The “AT₁ receptor” according to the invention may be present inits natural cellular environment and can be used together with thematerial associated with the receptor in its natural state, as well asin isolated form. With respect to its primary, secondary and tertiarystructures, the AT₁ receptor is well-known to those skilled in the art.Based on the weight of the whole receptor in the preparation to be usedaccording to the invention, the isolated receptor should account for atleast 0.5%, preferably at least 5%, more preferably at least 25%, and ina particularly preferred fashion at least 50%. The receptor ispreferably used in isolated form, i.e., essentially free of otherproteins, lipids, carbohydrates, or other substances naturallyassociated with the receptor. “Essentially free of” means that thereceptor is at least 75%, preferably at least 85%, more preferably atleast 95%, and especially preferably at least 99% free of otherproteins, lipids, carbohydrates, or other substances naturallyassociated with the receptor.

[0012] In connection with the present invention, the naturally occurringreceptor, as well as all modifications, mutants or derivatives of theAT₁ receptor can be used. Similarly, an AT₁ receptor produced by meansof recombinant techniques, which receptor includes amino acidmodifications such as inversions, deletions, insertions, additions,etc., can be used according to the invention, provided at least part ofthe essential function of the AT₁ receptor is present, namely, thecapability of binding antibodies. The AT₁ receptor being used may alsocomprise exceptional amino acids and/or modifications such asalkylation, oxidation, thiol modification, denaturation, oligomerizationand the like. The receptor can also be synthesized by chemical means.According to the invention, the AT₁ receptor particularly can be aprotein and/or a peptide, or a fusion protein which, in addition toother proteins, peptides or fragments thereof, includes the AT₁ receptoras a whole or in part. Using conventional methods, peptides orpolypeptides of the AT₁ receptor which have functionally analogousproperties can be determined by those skilled in the art. For example,such polypeptides or peptides have 50%, 60%, 70%, or 80%, preferably90%, more preferably 95%, and most preferably 98% homology to peptidesidentified as AT₁ receptor, and said homology can be determined e.g. bymeans of the Smith-Waterman homology search algorithm, using the MPSRCHprogram (Oxford Molecular), for example. The term “peptide of an AT₁receptor” used in the present invention comprises molecules differingfrom their original sequence by deletion(s), insertion(s),substitution(s) and/or other modifications well-known in the prior artor comprising a fragment of the original amino acid molecule, the AT₁receptor still exhibiting the properties mentioned above. Also includedare allele variants and modifications. Methods of producing the abovechanges in the amino acid sequence are well-known to those skilled inthe art and have been described in standard textbooks of MolecularBiology, e.g. in Sambrook et al., supra. Those skilled in the art willalso be able to determine whether an AT₁ receptor thus modified stillhas the properties mentioned above. Possible AT₁ receptor peptides usedaccording to the invention can be e.g. AVHYQSN, SHFYQTR or GYYFDTN. Inthe present specification, all of the above-illustrated modifications ofthe AT₁ receptor will be referred to as “functionally analogous peptidesor proteins” in brief.

[0013] “Biological materials” in the meaning of the invention can be allbiological tissues and fluids such as blood, lymph, urine, cerebralfluid. The biological material is collected from the patient andsubjected to the diagnosis according to the invention. Obviously, asample can also be treated or prepared for analysis using specificbiochemical and chemical means or methods.

[0014] The “auto-antibodies” in the meaning of the invention, which areto be detected, bind the AT₁ receptor in a specific fashion. Theauto-antibodies can also be modified antibodies (e.g. oligomeric,reduced, oxidized and labelled antibodies). The term auto-antibodiesused in the present specification comprises both intact molecules andauto-antibody fragments such as Fab, F(ab′)₂ and Fv capable of bindingspecific epitope determinants of the AT₁ receptor. In these fragmentsthe auto-antibody's capability of selectively binding its antigen orreceptor is retained in part, the fragments being defined as follows:

[0015] (1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be generated by cleavage of awhole antibody using the enzyme papain, thereby obtaining an intactlight chain and part of a heavy chain;

[0016] (2) the Fab′ fragment of an antibody molecule can be produced bytreatment of a whole antibody with pepsin and subsequent reduction,thereby obtaining an intact light chain and part of a heavy chain; twoFab′ fragments per antibody molecule are obtained;

[0017] (3) F(ab′)₂, the fragment of the antibody, which can be obtainedby treatment of a whole antibody with the enzyme pepsin withoutsubsequent reduction; F(ab′)₂ is a dimer comprised of two Fab′ fragmentsheld together by two disulfide bonds;

[0018] (4) Fv, defined as a fragment modified by genetic engineering,which includes the variable region of the light chain and the variableregion of the heavy chain and is expressed in the form of two chains;and

[0019] (5) single-chain antibody (SCA), defined as a molecule modifiedby genetic engineering, which includes the variable region of the lightchain and the variable region of the heavy chain linked by a suitablepolypeptide linker to form a genetically fused single-chain molecule.

[0020] The term epitope as used in the present invention represents anyantigen determinant on the AT₁ receptor. Epitope determinants normallyconsist of chemically active surface groups of molecules such as aminoacids or sugar side-chains and normally have specific features of thethree-dimensional structure, as well as specific charge properties.

[0021] The auto-antibody “binds specifically” to the AT₁ receptor, or,in doing so, shows specific immunoreactivity when the auto-antibodyassumes its function in a binding reaction in the presence of aheterogeneous population of AT₁ receptors or fragments thereof, therebyallowing a conclusion whether the AT₁ receptor or another biologicalstructure is present. Under the preset conditions of an immunoassay, theabove-mentioned auto-antibodies will preferably bind to a specificportion of the AT₁ receptor, while no significant binding to otherproteins present in the sample will take place.

[0022] “Patients” in the meaning of the invention are understood to beall persons, animals, plants, or microorganisms, irrespective of whetheror not they exhibit pathological changes. In the meaning of theinvention, any biological material collected from cells, tissues,organs, organisms or the like can be biological material of a patient tobe diagnosed.

[0023] An “immune reaction” in the meaning of the invention is aspecific interaction between the AT₁ receptor or peptides or proteins ofanalogous function and auto-antibodies. The immune reaction can bedetected using various immunoassays.

[0024] “Immunoassays” in the meaning of the invention are assaysutilizing the specific interaction between the AT₁ receptor and peptidesor proteins of analogous function and the auto-antibodies, in order todetect the presence or determine the concentration of theauto-antibodies. For example, the detection and quantification of theauto-antibodies can be performed with the aid of said peptides orproteins of analogous function, e.g. by immunoprecipitation orimmunoblotting. For example, immunoassays in the meaning of theinvention can be subdivided into the following steps:

[0025] 1) the auto-antibody/AT₁ receptor reaction,

[0026] 2) if required, separation of the auto-antibody/AT₁ receptorcomplex from other components of the reaction mixture, especially fromnon-bound auto-antibodies and AT₁ receptor, and

[0027] 3) measuring the response.

[0028] As for the auto-antibody/AT₁ receptor reaction, variousconfigurations are possible, e.g.

[0029] a) precipitation of one reactant with an excess of the other, or

[0030] b) competition between known quantities of auto-antibody or AT₁receptor and the material to be investigated.

[0031] For example, an assay for auto-antibodies can be performed by

[0032] a) using excess AT₁ receptors/peptides or proteins of analogousfunction, or

[0033] b) competition between a labelled auto-antibody of known amountand a non-labelled antibody of unknown amount for a defined quantity ofAT₁ receptor or of peptides or proteins of analogous function.

[0034] The AT₁ receptor can be immobilized on a solid support to allowseparation of the auto-antibody/AT₁ receptor complex. For example, thesolid support material can be nitrocellulose, polyvinyl chloride orpolystyrene, e.g. the well of a microtiter plate. To measure theauto-antibody/AT₁ receptor interaction, it is possible to use labelledauto-antibodies, labelled AT₁ receptors or secondary reagents, forexample. The AT₁ receptor can be labelled radioactively or with enzymesor with fluorescent compounds, for example. Irrespective of the labelthat is used, the response of the auto-antibody/AT₁ receptor interactioncan be enhanced by utilizing the affinity of the proteins avidin orstreptavidin for biotin. The immunoassays used according to theinvention can be:

[0035] 1) immunoassays using a radioactive label:

[0036] a) radioimmunoassays with competitive binding (RIA) and

[0037] b) immunoradiometric assay (IRMA),

[0038] 2) immunoassays using an enzyme label:

[0039] a) enzyme immunoassays (EIA) and

[0040] b) enzyme-linked immunosorbent assay (ELISA),

[0041] 3) immunoassays using a combination of radioisotope and enzymelabels (ultrasensitive enzyme radioimmunoassay (USERIA)).

[0042] In one embodiment of the invention, detection of human IgA and/orIgG auto-antibodies as auto-antibodies is envisaged. Auto-antibodies areglycoproteins, also referred to as immunoglobulins. Human antibodies canbe divided into five classes of immunoglobulins. Class A immunoglobulin(IgA) exists in a form which is dissolved in blood, as well as in asecretory variant. IgA comprises two basic classes. Secretory IgAconsists of two immunoglobulin basic molecules, together with a J chainand a secretory component. More specifically, IgA molecules can beprevalent in body secretions. Class IgG immunoglobulins represent themajor part among the immunoglobulins. The antibodies of the secondaryimmune response taking place upon contact of the immune system with aparticular antigen largely belong to the IgG class.

[0043] To allow a quantitative statement on the AT₁receptor/auto-antibody reaction, one of the reactants must be coupledwith a detectable label in such a way that the immunological propertiesof the components are largely retained, e.g. by means of thestreptavidin/biotin system or by using secondary antibodies.

[0044] In another preferred embodiment of the invention, animmunoprecipitation, a radioimmunoassay, an enzymatic assay, afluorescent immunoassay, a chemiluminescent immunoassay, a competitivebinding assay, an ELISA, and/or a homogeneous immunoassay can beemployed as immunoassay, preferably an ELISA. In immunoprecipitation,the reaction between the auto-antibody and the AT₁ receptor can beobserved in vitro, directly at the point of equilibrium where theconcentrations of both components coincide. At this point, animmunoprecipitation takes place. The immunoprecipitation can beperformed in semi-solid media such as agar gel. The diffusion of theimmunochemical components through such a gel generates a concentrationgradient which ensures that the conditions for immunoprecipitation aresatisfied at a particular point, provided the immune complexes willform. The position and nature of the immunoprecipitate in the gelfurnishes information on the concentrations of the immune components. Aqualitative evidence of an immune complex reaction can be obtained bydouble immunodiffusion. In the simplest form of immunoprecipitation, athin agarose gel is provided with two small wells receiving a smallamount of AT₁ receptor and the biological material containing theauto-antibodies. Following 24 hours of diffusion, the formation ofimmune complexes can be detected by a white line of precipitate betweenthe two wells. A more complex version is the Ochterlony assay whereinthe wells are arranged in the form of a circle on the gel plate, with anadditional well in the center of the circle. Various dilutions ofbiological material containing the auto-antibodies are placed in theouter wells, and the solution including the AT₁ receptor/peptides orproteins of analogous function is placed in the center of the circle.

[0045] Quantitative analysis is possible by simple immunodiffusion.While one component is distributed homogeneously in the gel, the otheris allowed to diffuse out of a well. For example, a solution includingthe AT₁ receptor/peptides of analogous function can be mixed into thegel, and a sample of biological material can be placed in the well,which material is to be investigated for an included auto-antibody byformation of a ring of precipitate.

[0046] Another possible assay to determine auto-antibodies is theradioimmunoassay (RIA). The RIA is a sensitive immunoassay based oncompetitive binding of an antigen to an antibody, wherein the amountbeing bound is determined quantitatively with the aid of radioactivelylabelled antigens. Advantageously, the RIA allows determination ofamounts of substances down to the picogram region.

[0047] Another assay for the determination of auto-antibodies is theenzyme immunoassay. This immunoassay is based on enzyme-labelling onereactant, e.g. the secondary antibody. Solid-phase systems such asvessels or pellets coated with antibody or antigen are advantageous,because removal thereof will not give rise to denaturation of proteins.Quantification of bound or free phase is effected by measuring theenzyme activity. For example, a colorless substrate can be added which,when converted by an enzyme, yields a colored product which can bedetected by spectroscopy and quantified using a standard sample. As arule, the substrate solution is added subsequent to separating bound andfree phases and, following a predetermined period of time, the reactionis quenched and the optical density of the product is determined,furnishing a measure for the amount of auto-antibodies present in thesample. The sensitivity of the enzyme reaction can be increased invarious ways. One method is the principle of enzymatic amplificationwherein the product of the first enzymatic reaction generates a closedcycle in a second enzyme system, resulting in a higher amount of coloredfinal product. One example of a closed enzymatic cycle is represented bythe enzyme alkaline phosphatase which hydrolyzes NADP⁺ to NAD⁺, thelatter entering an enzymatic cycle to form large amounts of a coloredproduct of nitrotetrazolium. Other enzymes can be peroxidase orβ-galactosidase. For example, luminol, β-galactosidase and p-nitrophenylphosphate are used as substrates. For example, detection of the boundauto-antibodies can be effected in a second incubation step using alabelled anti-auto-antibody. In a homogeneous immunoassay, theimmunocomplexes having formed are not removed. Another way of detectingthe auto-antibodies is by using a fluorescent immunoassay, withfluorescent labels constituting the basis of this assay. For example,the formation of immunocomplexes can be quantified via the label of AT₁receptor/functionally analogous peptides of the bound or free fractionin the immunochemical reaction mixture. For example, fluorescein orrhodamine can be used as fluorescence carriers, or labels based onchelate complexes of rare earths, such as organometallic coordinationcomplexes of europium.

[0048] Furthermore, the auto-antibodies can be detected in achemiluminescent immunoassay which, advantageously, is a simple andstable test system. Chemiluminescence is observed when a high-energychemical reaction produces molecules wherein the electrons are in anexcited state. Photons are emitted when these excited molecules returnto their ground state. For example, such chemiluminescent molecules canbe coupled to AT₁ receptors or peptides of analogous function and usedin an immunoassay. For example, the AT₁ receptors/functionally analogouspeptides can be labelled with luminol or acridinium salts. Acridiniumsalts undergo chemiluminescent reactions without requiring a catalyst,as is the case with luminol derivatives. The catalysts comprise a widespectrum of substances ranging from simple transition metal cations upto complex enzymes. Advantageously, acridinium salts merely requireaddition of a dilute alkaline hydrogen peroxide solution to trigger achemiluminescent reaction.

[0049] It is also possible to detect the auto-antibodies in acompetitive binding assay wherein limited amounts of labelled AT₁receptor or functionally analogous peptide competing for theauto-antibody are employed. Such a method can be advantageous in thosecases where purified AT₁ receptors or functionally analogous peptidesare available for labelling. For example, an AT₁ receptor bound to asolid phase can be supplied, to which receptor an auto-antibody presentin the sample can bind.

[0050] The invention also relates to the use of the anti AT₁ receptor orof functionally analogous peptides or proteins in order to predict therisk of transplant rejection. To this end, the auto-antibodies aredetected in the above-described immunoassays in a per se known manner.

[0051] The invention also relates to an immunological test kit includingthe AT₁ receptor or peptides or proteins of analogous function. The testkit of the invention comprises at least one complete AT₁ receptor orfunctionally analogous peptides or proteins of said receptor, optionallybound to a solid phase. Furthermore, the test kit may also comprisebuffers, a specific conjugate together with an enzyme, a wash solution,a substrate solution to detect the immune reaction, and/or a quenchingsolution. Using these substances, a person skilled in the art will beable to perform e.g. an ELISA to detect the auto-antibodies. Thebuffers, specific conjugate plus enzyme, wash solution, substratesolution to detect the immune reaction, and quenching solution arewell-known to those skilled in the art. For example, it would besufficient to have the test kit comprise a freeze-dried AT₁ receptor orpeptides or proteins of AT₁-analogous function and to add the buffersand other solutions immediately prior to testing the biologicalmaterial. However, it is also possible to provide the test kit with theAT₁ receptor or its functionally analogous peptides or proteins bound toa solid phase. To detect the auto-antibodies, the specific conjugate,wash solution, substrate solution, and quenching solution, which can becomponents of the test kit, have to be added according to a modewell-known to those skilled in the art.

[0052] In another advantageous embodiment of the invention, it isenvisaged that the test kit is a test strip comprising the AT₁ receptoror its functionally analogous peptides or proteins immobilized on asolid phase. For example, the test strip can be immersed in serum orother biological samples and incubated. Using a specific biochemicalreaction on the test strip after formation of the AT₁receptor/auto-antibody complex, a specific color reaction can betriggered, by means of which the auto-antibodies can be detected.

[0053] The test system of the invention permits quantification ofanti-AT₁ receptor/auto-antibodies directly in a biological material,e.g. in plasma of patients. The detection method according to theinvention is timesaving and cost-effective. Large amounts of samples canbe tested and, owing to the low amount of equipment required, routinelaboratories can be used.

[0054] Without intending to be limiting, the invention will beillustrated with reference to the following example.

EXAMPLE AT₁ Elisa

[0055] A suitable streptavidin-coated microtiter plate is loaded withthe biotinylated peptide SAFHYESQNSTL. To this end, 100 μl of a solutionper well on the microtiter plate is incubated with 5 μg/ml in a suitabledilution buffer. To measure the non-specific binding, wells are alsofilled with 100 μl of dilution buffer only.

[0056] Subsequent to the time period of reaction, the peptide solutionis removed by decanting, and each well is washed three times with about250 μl of a suitable wash buffer.

[0057] Thereafter, 100 μl/well of sera diluted in dilution buffer areplaced on both the peptide-loaded and comparative plates and incubated.Subsequently, the wells are washed as described above.

[0058] The bound antibodies are detected using a goat-anti-humanimmunoglobulin G antibody having peroxidase coupled thereto. To thisend, the antibody is diluted in dilution buffer and incubated (100μl/well), and this is followed by three wash steps (see above).

[0059] Following addition of 100 μl of a ready-for-use substratesolution (e.g. 3,3′,5,5′-tetramethylbenzidine), a color developsdepending on the amount of peroxidase in the well. The substratereaction is terminated by addition of 100 μl of 0.5 M sulfuric acid. Theabsorption is measured at 450 nm.

[0060] For evaluation, the difference between the absorptions ofpeptide-loaded microtiter plate and comparative plate with no peptide isformed. Samples having higher absorption than the cut-off are positive.The cut-off is calculated from the mean value of the absorption ofnegative donors plus three times the standard deviation. In general, acut-off control or a standard dilution series allowing quantification inrelative units is co-performed in the test.

1. A method of predicting the risk of transplant rejection,characterized in that anti-AT₁ receptor auto-antibodies are detected inbiological materials of a patient to be diagnosed, a risk of transplantrejection being present if auto-antibodies are detected.
 2. The methodaccording to claim 1, characterized in that detection is effected bymeans of an immune reaction using the AT₁, receptor or functionallyanalogous peptides or proteins.
 3. The method according to claim 1 or 2,characterized in that human IgA and/or IgG auto-antibodies are detected.4. The method according to claim 1 any of claims 1 to 3, characterizedin that detection of the auto-antibodies is effected in an immunoassay.5. The method according to claim 4, characterized in that animmunoprecipitation, a radioimmunoassay, an enzyme immunoassay, afluorescent immunoassay, a chemiluminescent immunoassay, a competitivebinding assay, an ELISA, or a homogeneous immunoassay is used asimmunoassay, preferably an ELISA.
 6. Use of the AT₁ receptor orfunctionally analogous peptides or proteins to predict the risk oftransplant rejection.
 7. Immunological test kit for predicting the riskof transplant rejection, which test kit comprises said AT₁, receptor orfunctionally analogous peptides or proteins thereof.
 8. The test kitaccording to claim 7, characterized in that the test kit comprises atest strip having the AT₁, receptor or functionally analogous peptidesor proteins thereof immobilized thereon.